EP1357190B1 - Methods and markers for simple transformation and selection of recombinant ciliates - Google Patents

Description

The present invention relates to a process for the production of genetically modified (recombinant) ciliates without the use of negative selection markers and to an efficient process for the production of proteins by means of the so-modified protists.

The production of recombinant proteins by heterologous protein expression represents an alternative to the recovery of proteins from natural sources. Natural sources of proteins that serve, for example, as pharmaceuticals are often limited, very expensive to purify, or simply unavailable. In addition, they can be very problematic due to the risk of toxic or especially infectious contaminants. On the other hand, biotechnology today enables the economical and safe production of a whole range of proteins in sufficient quantities by means of heterologous expression for a wide variety of applications: eg antibodies (for diagnosis, passive immunization and research), hormones (such as insulin, erythropoietin (EPO), Interleukins, etc. for therapeutic use), enzymes (eg for use in food technology, diagnosis, research), blood factors (for the treatment of hemophilia), vaccines, etc. ( Glick & Pasternak 1998, Molecular Biotechnology, ASM Press, Washington DC, Chapter 10: 227-252 ).

Proteins for medical use, especially human proteins, must be identical in their biochemical, biophysical and functional properties to the natural protein. In a recombinant production of such proteins by heterologous gene expression is to be noted that there are a number of post-translational protein modifications in eukaryotic cells as opposed to bacteria: formation of disulfide bridges, proteolytic cleavage of precursor proteins, modifications of amino acid residues (phosphorylation, acetylation, acylation, sulfation , Carboxylation, myristylation, palmitylation and especially glycosylations). In addition, proteins in eukaryotic cells often become first brought into the correct three-dimensional structure through a complex mechanism involving chaperones.

These modifications play a very important role in the specific structural and functional properties of the proteins, such as the activity of enzymes, the specificity (receptor binding, cell recognition), folding, solubility etc. of the protein ( Ashford & Platt 1998, in: Post-Translational Processing - A Practical Approach, Ed. Higgins & Hames, Oxford University Press, Chapter 4: 135-174 ; Glick & Pasternak 1998, Molecular Biotechnology, ASM Press, Washington DC, Chapter 7: 145-169 ).

Deviations from the natural structure, for example, may lead to inactivation of the proteins or have a high allergenic potential.

Although a whole range of bacterial and eukaryotic expression systems have been established for the production of recombinant proteins, there is no universal system which covers the entire spectrum of possible protein modifications-in particular in the case of eukaryotic proteins-and thus would be universally applicable ( Castillo 1995, Bioprocess Technology 21: 13-45 ; Geise et al. 1996, Prot. Expr. Purif. 8: 271-282 ; Verma et al. 1998 J. Immunological Methods 216: 165-181 ; Glick & Pasternak 1998, Molecular Biotechnology, ASM Press, Washington DC, Chapter 7: 145-169 ). Another problem is that some of the widely used systems introduce unusual and sometimes unwanted post translational protein modifications. Thus, recombinantly expressed proteins from yeasts are sometimes extremely strongly modified with mannose residues. These so-called "high mannose" structures of yeasts consist of about 8-50 mannose residues and thus differ considerably from the mannose-rich glycoprotein structures from mammalian cells which have a maximum of 5-9 mannose residues ( Moremen et al. 1994, Glycobiology 4 (2): 113-125 ). These yeast-typical mannose structures are strong allergens and thus very useful in the production of recombinant glycoproteins for therapeutic use problematic ( Tuite et al. 1999, in Protein Expression - A Practical Approach, Ed. Higgins & Hames, Oxford University Press, Chapter 3: especially page 76 ). In addition, no hybrid or complex glycoprotein structures can be formed in yeasts, further limiting their use as an expression system.

In contrast, plants that have recently been discussed and used more frequently as production systems for recombinant proteins have xylose on the glycoprotein structures instead of mammalian sialic acid (Ashford & Platt, supra). Xylose and the α-1,3-linked fucose also found in plants can pose an allergic risk and are therefore problematic ( Jenkins et al. 1996, Nature Biotech. 14: 975-981 ).

Therefore, there is a great need for new, alternative eukaryotic expression systems, especially as an alternative to the very costly and expensive production of recombinant proteins using mammalian cells.

Such a system should ideally meet the following requirements: 1) Selection markers and regulatory DNA elements (such as transcriptional and translational signals, etc.) must be available. 2) The expression system should enable important eukaryotic post-translational protein modifications and 3) the production of the recombinant proteins should be as simple and economical as possible, e.g. by the possibility of fermentation of the cells or organisms on a production scale (for example, several 1000 L) on simple media and simple workup of the products.

To the already established eukaryotic expression systems, such as yeast mammalian or insect culture cells, protozoa or protists (as definition see eg Henderson's Dictionary of biological terms, 10th ed. 1989, Eleanor Lawrence, Longman Scientific & Technical, Engl and, or Margulis et al. (Eds) 1990. Handbook of Protoctista, Jones & Bartlett, Bost one; van den Hoek et al. 1995, Algae - An introduction to phycology, Cambridge University Press ) represent an interesting alternative. These organisms are one very heterogeneous group of eukaryotic, usually single-celled microorganisms. They have the compartmentation and differentiation typical of eukaryotic cells. Some are relatively closely related to higher eukaryotes but, on the other hand, are more similar to yeasts or even bacteria in terms of culture and growth, and relatively easily ferment at high cell density on simple large scale media.

A protist of interest for the expression of heterologous proteins is, for example, the ciliate Tetrahymena , in particular Tetrahymena thermophila . It is a non-pathogenic, unicellular, eukaryotic microorganism that is relatively closely related to higher eukaryotes and has the typical cell differentiation. The posttranslational protein modifications in Tetrahymena are more similar to those in mammalian cells than, for example, those detected in yeast or other eukaryotic expression systems. For example, in Tetrahymena, no strong antigenic sugar chains are found on glycoproteins, such as in yeast ("high mannose" structures), and plant or lower animal cell culture-based expression systems (xylose residues) (see above). Although Tetrahymena is a true, complexly differentiated eukaryote, it is more similar to simple yeasts or bacteria in its culturing and growth characteristics, and can be easily fermented on relatively cheap skim milk media on a large scale. Under optimal conditions, the generation time is approximately 1.5-3 h and very high cell densities (2.2 × 10 ⁷ cells / ml, corresponding to 48 g / l of dry biomass) can be achieved ( Kiy and Tiedke 1992, Appl. Microbiol. Biotechnol. 37: 576-579 ; Kiy and Tiedke 1992, Appl. Microbiol. Biotechnol. 38: 141-146 ). This makes Tetrahymena very attractive for large-scale fermentative production of recombinant proteins.

Another advantageous aspect of Tetrahymena as expression system is the fact that in Tetrahymena an integration of the heterologous gene by homologous DNA recombination is possible. As a result, mitotically stable transformants can be generated. In addition, homologous DNA recombination also enables targeted gene knock-outs ( Bruns & Cassidy-Hanley in: Methods in Cell Biology, Volume 62, Ed. Asai & Forney, Academic Press (1999) 501-512 ; Hai et al. in: Methods in Cell Biology, Volume 62, Ed. Asai & Forney, Academic Press (1999) 514-531 ; Gaertig et al. (1999) Nature Biotech. 17: 462-465 or Cassidy-Hanley et al. 1997 Genetics 146: 135-147 ). In addition, either the somatic macronucleus or the generative micronucleus can be transformed. In Makronucleustransformation obtained sterile transformants, which may be advantageous for security or acceptance issues.

The transformation of Tetrahymena can be achieved by microinjection, electroporation or microparticle bombardment. There are a number of vectors, promoters, etc. available. The selection of the transformants is usually carried out by means of a resistance marker. So Tetrahymena z. B. successfully transformed with a rDNA vector. Selection was made in this case by a paromomycin resistance mutation of the rRNA ( Tondravi et al. 1986, PNAS 83: 4396 ; Yu et al. 1989, PNAS 86: 8487-8491 ). In further transformation experiments, cycloheximide or neomycin resistances were successfully expressed in Tetrahymena ( Yao et al. 1991, PNAS 88: 9493-9497 ; Kahn et al. 1993, PNAS 90: 9295-9299 ). In addition to these marker genes have Gaertig et al. (1999, Nature Biotech. 17: 462-465 ) successfully expressed two recombinant proteins in Tetrahymena (a fish parasite antigen and a chicken partial ovalbumin). The selection was carried out with the aid of paclitaxel (taxol). This by Gaertig et al. developed system is patent pending ( WO 00/46381 ).

Only a few other protists or protozoans have been described for transformation and heterologous protein expression. Another ciliate is paramecium ( Boileau et al. 1999, J. Eukaryot. Microbiol. 46: 56-65 ) mentioned. However, various attempts have also been made to transform and express recombinant proteins in parasitic protozoa, such as Trypanosoma, Leishmania, Plasmodium and others (Beverly 2000, WO 00/58483 ). An overview of it gives Kelly (1997, Advances in Parasitology, Vol. 39, 227-270 ). One possibility for heterologous protein expression was also shown in the slime mold Dictyostelium discoideum ( Manstein et al. 1995, Gene 162: 129-134 . Jung and Williams 1997, Biotechnol. Appl. Biochem. 25: 3-8 ) but also in photoautotrophic protists (microalgae) like Chlamydomonas ( Hall et al. 1993, Gene 124: 75-81 ), Volvox ( Schiedlmeier et al. 1994, PNAS 91: 5080-5084 ), certain dinoflagellates ( Lohuis & Miller 1998, Plant Journal 13: 427-435 ) and diatoms ( Dunahay et al. 1995, J. Phycol. 31: 1004-1012 ). In most cases, however, only simple resistance markers or non-human selection markers were expressed.

None of these organisms has been used on a larger scale for the production of recombinant proteins. A major problem for many of these and other potentially interesting systems is the lack of well-established genetic engineering techniques and, in particular, molecular biological "tools" such as vectors, markers, etc.

The presence of a selective marker is a necessary prerequisite for genetically modifying cells. In general, the selection of transformed cells or organisms by negative selection marker, usually the resistance to an antibiotic (eg, ampicillin, kanamycin, tetracycline, neomycin, etc.). Rarely, selection is made by introducing an essential gene into a defective cell type that affects this gene product (positive selection or selection by complementation). These include, for example, LEU- or URA3-based selection systems in yeasts (see Glick & Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, 1998, 2nd ed., ASM Press, Washington, DC pages 109-169 ). However, this second approach is not available for the little-studied "new organisms" such as the already mentioned protists. As a result, the usability of these systems, especially for the production of recombinant proteins on a production scale, severely limited.

Negative selection generally has serious disadvantages. On the one hand, unnecessary DNA has to be added to the production organism for the desired product which may raise concerns about biosafety but, above all, public acceptance. On the other hand, the organisms have to be cultivated in the presence of the corresponding antibiotics during the entire production time in order to maintain the selection pressure, which in turn enormously increases the costs. It is not only the costs of the antibiotic itself, or the disposal of production waste (media, ...) to be considered, but also the processing of the proteins can be much more difficult. But apart from cost-effectiveness, environmental compatibility, biological or genetic engineering safety and, above all, public acceptance are very problematic. In addition, problems of a purely technical nature arise when the organism has to be transformed several times. The simultaneous presence of several antibiotics or the simultaneous expression of several antibiotic resistance genes may, if at all possible, have an extremely adverse effect on the organisms or lead to unexpected side effects.

In view of the prior art, it was therefore an object of the present invention to provide a method for the positive selection of genetically modified protists, which, inter alia, allows efficient production of recombinant proteins.

This is solved, as well as other tasks not explicitly mentioned, but which are readily derivable or deducible from the contexts discussed hereinbelow by the embodiments of the present invention as defined in the claims.

A method for the production of recombinant ciliates can be achieved in a surprisingly simple manner by preparing an auxotrophic mutant of the ciliate, then transforming this mutant with recombinant DNA which contains at least one gene for complementation of the corresponding auxotrophy, and finally the resulting recombinant ciliates on a minimal medium, which allows growth only in accordance with complemented ciliates, Ciliates of the genera Paramecium and Tetrahymena are used. Especially For this method, in order to permanently maintain the selection pressure, ultimately neither a selection for a resistance against an antibiotic nor even the presence of undesired heterologous genes in the recombinant organism is necessary. An addition of antibiotics to the culture medium can thus be omitted. The method can also be easily applied several times to the same organism strain, transforming it with a variety of desired recombinant genes, without any (over) expression of further ev. Unwanted genes.

In another aspect of the present invention, a method of producing recombinant proteins can be provided in an equally simple manner by preparing recombinant ciliates as described above, the recombinant DNA additionally comprising at least one functional recombinant gene for transforming the ciliates a protein to be expressed. The recombinant ciliates are subsequently cultivated so that the proteins can be expressed and subsequently isolated.

In a preferred embodiment of the present invention, the production of the auxotrophic mutant is accomplished by knock-out of an essential gene. The knock-out can be achieved by complete deletion of the corresponding gene or by mutation of the same. Mutations are, for example, insertions, deletions, inversions or even the exchange of individual base pairs. Gene deletions or mutations can be introduced into the target organism by methods known to those skilled in the art. Inter alia, here in vitro mutagenesis, for example, by inaccurate PCR (error-prone PCR) or about the Kunkel method ( Sambrook et al. Molecular Cloning, A Laboratory Manual, Coldspring Harbor, NY ), or exon-shuffling or gene site saturation mutagenization (GSSM) (see eg: www.Diversa.com and www.Maxygen.com)

For the purposes of the present invention, essential genes for the production of an auxotrophic knock-out mutant are understood to be essential metabolic genes, e.g. fatty acid, sterol, amino acid biosynthesis, etc., whose failure can be compensated for by adding the appropriate molecules (fatty acids, sterols, amino acids, etc.) into the culture medium (also called marker). By targeted knock-out of such genes, the cells become auxotrophic for products of this metabolic pathway. In the present invention, this is exemplified for sterol and fatty acid biosynthesis. By adding the appropriate metabolite, i. Cholesterol, or fatty acids, the cells can survive this knock-out. Without the additives, the cells die quickly.

Genes encoding, for example, a triterpenoid cyclase (synonym for tetrahymanol cyclase), a delta-6-desaturase or a delta-9-desaturase are therefore, according to the invention, suitable targets for a knockout to be an auxotrophic mutant of the respective organism manufacture.

A preferred gene for a triterpenoid cyclase according to the invention was disclosed in the German patent application DE 199 57 889 A1 described. A preferred gene for a delta-6-desaturase according to the invention was disclosed in the German patent application DE 100 44 468 A1 described. A preferred gene for a delta-9-desaturase according to the invention was obtained from Nakashima S. et al. m Biochem. J. 317, 29-34 (1996 ), the respective sequence can be found in the GenBank under Accession No .: EMBL D83478. See GenBank Benson, DA et al., Nuc. Acid Res., 28 (1), 15-18 (2000 ).

According to the invention, by reintroducing the switched-off gene, the cells regain their autotrophy for this metabolite. In In this case it is said that the auxotrophy is complemented. The selection for successfully transformed cells is carried out in minimal medium, ie in particular omitting the metabolite for which the non-successfully transformed organisms are auxotrophic. According to the invention, a minimal medium is to be understood as a medium which contains all the necessary building blocks which make it possible to survive the cells (carbon source (s), such as, for example, sugar, nitrogen source (s), possibly amino acids, vitamins, trace elements, etc.) just does not contain that metabolite for which the parent organism is auxotrophic.

When this gene, which complements the auxotrophy, is coupled to a gene coding for a heterologous protein, the transformants can be identified without the addition of a selection marker, and moreover the successful stable expression is not dependent on the addition of a selection marker, as described e.g. B. is typically the case with recombinant E. coli . As a further positive effect, apart from the target gene to be expressed, no foreign DNA is introduced into the cell. In addition, in organisms in which homologous recombination occurs (eg, Tetrahymena ), the DNA is not randomly integrated into the genome but at a certain position, namely the natural position of the marker gene.

However, it is clear to the person skilled in the art that auxotrophy can also be complemented by means of corresponding heterologous or in vitro modified genes.

According to the invention suitable for selection by the method described above, transformed ciliates, the genera Paramecium or Tetrahymena , particularly preferably the species Tetrahymena thermophila .

Recombinant DNA for the transformation of the auxotrophic protist mutant can, for. B. be a vector, ie any kind of nucleic acids, such as. A plasmid, cosmid, virus, an autonomously replicating sequence, a phage, a linear or circular, single or double stranded DNA or RNA molecule which is in the target organism can replicate itself or can be incorporated into the genome, but at least in the target organism contains functional sequences.

According to the invention, functional DNA sequences are understood as meaning those DNA segments which can fulfill their respective function in the recombinant organism.

For the purposes of the present invention, a functional gene is understood as meaning, for example, a gene which can be expressed in the target organism. In particular, a functional gene therefore comprises, in addition to a coding sequence, a promoter functional in the target organism, which leads to the transcription of the coding sequence. Such a functional promoter may include , but not limited to, one or more TATA boxes, CCAAT boxes, GC boxes or enhancer sequences. Furthermore, the functional gene may include a functional terminator in the target organism which leads to the termination of the transcription and contains signal sequences that lead to the polyadenylation of the mRNA. The coding sequence of the functional gene also has all the properties necessary for translation in the target organism (for example, start codon (eg ATG), stop codon (eg TGA, in particular Tetrahymena ) A-rich regions before start (Translation initiation sites ), Kozak sequences, poly A site The gene may also have the specific recombinant organism codonusage (for Tetrahymena see, for example Wuitschick & Karrer, J. Eukaryot. Microbiol. (1999 )).

According to the invention, a recombinant gene for a protein to be expressed in a recombinant ciliate in a process for producing recombinant proteins is a homologous or a heterologous gene. If it is a heterologous gene, it is preferably isolated from a vertebrate, more preferably from humans. A preferred example of this is human erythropoietin. Further recombinant genes which are preferred according to the invention for proteins to be expressed in recombinant ciliates are those from organisms which are capable of inducing diseases in humans or in animals (such as, for example: malaria), with the aid of recombinant proteins or also the biomass containing the recombinant proteins or parts thereof, to be able to achieve an active immunization.

• Abb. 1: Struktur des Tetrahymanol-Gens pgTHC
• Abb. 2: Tetrahymanol-Knock-out Konstrukt pgTHC::neo
• Abb. 3: Expressionskonstrukt pBTHC
• Abb. 4: Struktur des Tetrahymanol-Cyclase/neo Konstruktes pgTHC+neo
• Abb. 5: Delta-6 Desaturase Knock-out Konstrukt pgDES6::neo
• Abb. 6: Struktur des genomischen delta-6 Desaturase-Gens pgDES6
• Abb. 7: Struktur des delta-6 Desaturase/neo Konstruktes pgDES6+neo

Description of the pictures:

• Fig. 1 : Structure of the Tetrahymanol gene pgTHC
• Fig. 2 : Tetrahymanol knock-out construct pgTHC :: neo
• Fig. 3 : Expression construct pBTHC
• Fig. 4 : Structure of the tetrahymanol cyclase / neo construct pgTHC + neo
• Fig. 5 : Delta-6 desaturase knock-out construct pgDES6 :: neo
• Fig. 6 : Structure of the genomic delta-6 desaturase gene pgDES6
• Fig. 7 : Structure of delta-6 desaturase / neo construct pgDES6 + neo

The following examples serve to illustrate the invention, without limiting the invention to these examples.

EXAMPLE 1: Organisms and Culture Conditions

Tetrahymena thermophila (strains B1868 VII, B2086 II, B * VI, CU428, CU427, CU522, provided by Dr. J. Gaertig, University of Georgia, Athens, GA, USA) were prepared in modified SPP medium (2% proteose peptone , 0.1% yeast extract, 0.2% glucose, 0.003% Fe-EDTA (Gaertig et al. (1994) PNAS 91: 4549-4553)) or skimmed milk medium (2% skimmed milk powder, 0.5% yeast extract, 1% Glucose, 0.003% Fe-EDTA) or MYG medium (2% skimmed milk powder, 0.1% yeast extract, 0.2% glucose, 0.003% Fe-EDTA) with addition of antibiotic solution (100 U / ml penicillin, 100 μg / ml streptomycin and 0.25 μg / ml amphotericin B (SPPA medium)) at 30 ° C in 50 ml volume in 250 ml Erlenmeyer flasks with shaking (150 rev / min) cultured.

Plasmids and phages were propagated and selected in E. coli XL1-Blue MRF ', TOP10F' or JM109 (Stratagene, Invitrogen, GibcoBRL Life Technologies). The bacteria were cultivated under standard conditions in LB or NZY medium, with antibiotics in standard concentrations ( Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, New York ).

EXAMPLE 2: Preparation of Triterpenoid Cyclase Knockout Construct

For the production of the knock-out construct was in the genomic sequence of triterpenoid-cyclase (patent application DE 199 57 889 A1 ) a neo-cassette from the plasmid p4T2-1ΔH3 ( Gaertig et al. (1994) Nucl. Acids Res. 22: 5391-5398 ) advertised. It is the neomycin resistance gene under the control of the Tetrahymena histone H4 promoter and the 3 'flanking sequence of the BTU2 gene. This construct mediates resistance to paromomycin in Tetrahymena . The plasmid p4T2-1ΔH3 was cut with Eco RV / Sma I and the approximately 1.4 kb fragment comprising the neo cassette was ligated into the Eco RV cut plasmid pgTHC into the genomic sequence of Tetrahymena triterpenoid cyclase. As a result, the plasmid pgTHC :: neo was generated (s. Fig. 2 ). In a successful transformation, the construct for triterpenoid cyclase was replaced by homologous recombination by this construct, thereby conferring resistance of the cells to paromomycin.

EXAMPLE 3: Production of the Expression Construct pBTHC

• THC-Nsi-F: 5'- CTCTTTCATACATGCATAAGATACTCATAGGC-3' (SEQ ID No 1) und
• THC-Bam-R: 5'- GGCTTGGATCCTCAAATATTTTATTTTTATACAGG-3' (SEQ ID No 2)

erzeugten PCR-Produkte, welche die vollständige kodierende Sequenz der Tetrahymanol-Cyclase, flankiert von Nsi I und Bam HI Schnittstellen, enthielten. Die PCR-Produkte und das Plasmid pBICH3-Nsi wurden mit den Restriktionsenzymen Nsi I und Bam HI geschnitten, über ein Agarosegel gereinigt und ligiert (s. Abb. 3). Das so entstandene Expressionskonstrukt pBTHC enthielt die vollständige die Triterpenoid-Cyclase kodierende Sequenz inseriert in einem korrekten Leseraster in die regulatorischen Sequenzen des BTU1 Gens. Für die Transformation von Tetrahymena wurden die Konstrukte durch einen Verdau mit den Restriktionsenzymen Xba I und Sal I linearisiert.The vector pBICH3 ( Gaertig et al. 1999 Nature Biotech. 17: 462-465 . WO 00/46381 ) contains the coding sequence of the Ichthyophthirius I antigen (G1) preprotein flanked by the non-coding, regulatory sequences of the Tetrahymena thermophila BTU1 gene. A modified plasmid (pBICH3-Nsi) with an Nsi I interface at the start (provided by J. Gaertig, University of Georgia, Athens, GA, USA) was used to prepare the tetrahymanol cyclase expression construct pBTHC. For this purpose, by means of PCR Nsi I and Bam HI cleavage sites at the start and stop of the coding sequences of Tetrahymanol cyclase of Tetrahymena were inserted. For the PCR, isolated plasmids containing the complete cDNA sequences of the tetrahymanol cyclase (pTHC) were used as template. The primers

• THC-Nsi-F: 5'-CTCTTTCATACATGCATAAGATACTCATAGGC-3 '(SEQ ID No 1) and
• THC-Bam-R: 5'-GGCTTGGATCCTCAAATATTTTATTTTTATACAGG-3 '(SEQ ID No 2)

generated PCR products containing the complete coding sequence of the tetrahymanol cyclase flanked by Nsi I and Bam HI cleavage sites. The PCR products and the plasmid pBICH3-Nsi were cut with the restriction enzymes Nsi I and Bam HI, purified on an agarose gel and ligated (s. Fig. 3 ). The resulting expression construct pBTHC contained the complete triterpenoid cyclase coding sequence inserted in a correct reading frame into the regulatory sequences of the BTU1 gene. For the transformation of Tetrahymena , the constructs were linearized by digestion with the restriction enzymes Xba I and Sal I.

In a successful transformation, homologous recombination replaced the BTU1 gene with these constructs, conferring cell resistance to paclitaxel.

EXAMPLE 4: Macronucleus Transformation of Tetrahymena with the Tetrahymanol Cyclase Expression Construct pBTHC

For a transformation, 5 × 10 ⁶ Tetrahymena thermophila cells (CU522) were used. The cells were cultivated in 50 ml of SPPA medium at 30 ° C. in a 250 ml Erlenmeyer flask on a shaker at 150 rpm to a cell density of about 3-5 × 10 ⁵ cells / ml. The cells were through Centrifugation (1200 g) for 5 min and the cell pellet was resuspended in 50 ml of 10 mM Tris-HCl (pH 7.5) and centrifuged as before. This washing step was repeated and the cells resuspended in 10 mM Tris-HCl (pH 7.5, plus antibiotics) at a cell density of 3 x 10 ⁵ cells / ml, transferred to a 250 ml Erlenmeyer flask and shaken for 30-60 h without shaking ° C incubated (starvation phase). After the starvation phase, the cell count was again determined, centrifuged as above, and the cells were adjusted with 10 mM Tris-HCl (pH 7.5) to a concentration of 5 × 10 ⁶ cells / ml. One ml of the cell suspension was used for the transformation. Transformation was by microparticle bombardment (see below). For regeneration, the cells were taken up in SSPA medium and incubated at 30 ° C without shaking in the Erlenmeyer flask. After 3 h, Paclitaxel® was added at a final concentration of 20 μM and the cells transferred in 100 μl aliquots to 96-well microtiter plates. The cells were incubated in a moist, darkened box at 30 ° C. After 2-3 days, paclitaxel-resistant clones could be identified. Positive clones were inoculated into fresh medium with 25 μM paclitaxel. By cultivating the cells in increasing paclitaxel concentration (up to 80 μM), a complete "phenotypic assortment" (Gaertig & Kapler (1999)) was achieved.

For analysis of the clones, about 4 ml cultures were grown in SPPA with paclitaxel, DNA isolated ( Jacek Gaertig et al. (1994) PNAS 91: 4549-4553 ) and amplified by PCR the integrated into the BTU1 locus DNA. The primers used were the BTU1-specific primers

BTUI-5'F (AAAAATAAAAAGTTTGAAAAAAACCTTC (SEQ ID No 3), ca 50 bp before the start codon and BTU1-3'R (GTTTAGCTGACCGATTCAGTTC (SEQ ID No 4), 3 bp behind the stop codon PCR products were uncut and Hind III, Sac I or Pst I, cut on a 1% agarose gel, and complete phenotypic assortment was checked by RT-PCR with the BTU1-specific primers (Gaertig & Kapler (1999)).

EXAMPLE 5: Preparation of the delta-6-desaturase knock-out construct pgDES6 :: neo

For the production of the knock-out construct, a neo-cassette from the plasmid p4T2-1ΔH3 (patent application DE 100 44 468 A1 ) advertised. It is the neomycin resistance gene under the control of the Tetrahymena histone H4 promoter and the 3 'flanking sequence of the BTU2 gene. This construct mediates resistance to paromomycin in Tetrahymena . The plasmid P4T2-1ΔH3 was cut with Eco RV / Sma I and the approximately 1.4 kb fragment of the neo-cassette was inserted into the Eco RV cut plasmid pgDES6 (patent application DE 100 44 468 A1 ) are ligated into the genomic sequence of Tetrahymena delta-6-desaturase (s. Fig. 5 ). This generated the plasmid pgDES6 :: neo. In a successful transformation, the gene for delta-6-desaturase was replaced by homologous recombination by this construct, conferring resistance of the cells to paromomycin.

EXAMPLE 6: Micronucleus and Macronucleus Transformation of Tetrahymena with the Knock Out Constructs pgTHC :: neo and pgDES6 :: neo

Tetrahymena strains of different mating type (CU428 VII and B2086 II) were cultured separately in SPPA medium at 30 ° C with shaking (150 rpm) in Erlenmeyer flasks. At a cell density of 3-5 x 10 ⁵ cells / ml, the cells were centrifuged for 5 min at room temperature (1200 g). The cells were washed three times with 50 ml of 10 mM Tris-HCl (pH 7.5) and finally resuspended in 50 ml of 10 mM Tris-HCl (pH 7.5) and mixed with antibiotic solution and then shaken in Erlenmeyer flasks at 30 ° C incubated. After about 4 h, the cell count of both cultures was determined again and adjusted with 10 mM Tris-HCl (pH 7.5) to 3 x 10 ⁵ cells / ml. Thereafter, the cultures were incubated for a further 16-20 h at 30 ° C. After this starvation phase, the same (absolute) cell number was mixed by both cultures in a 2-L Erlenmeyer flask. The Cells were incubated at 30 ° C (initiation of conjugation) and after 2 h the efficiency of conjugation was determined. For a successful transformation about 30% of the cells should be present as pairs at this time.

For the micronucleus transformation, 1 × 10 ⁷ conjugating cells (5 × 10 ⁶ pairs) were centrifuged for 5 min at 1200 g for 5 h, 3.5 h, 4 h and 4.5 h after the beginning of the conjugation, and the cell pellet in 1 10 mM Tris-HCl (pH 7.5).

For the transformation of the new Macronucleus plants, cells were centrifuged 11 hours after the start of conjugation as above and resuspended in Tris-HCl.

The transformation was carried out by microparticle bombardment (see below).

For the cultivation of the tetrahymanol cyclase knock-out mutants, 10 μg / ml cholesterol was added to the medium.

For the cultivation of the delta-6-desaturase knockout mutants, 200 μg / ml borage oil (20-25% GLA, SIGMA) was added to the medium.

By selecting for paromomycin resistance, transformed cells could be identified. In the transformation of the micronucleus, paromomycin (100 μg / ml final concentration) was added 11 hours after the beginning of the conjugation and the cells were distributed in aliquots of 100 μl on 96-well microtiter plates. The cells were incubated in a humid box at 30 ° C. After 2-3 days, resistant clones could be identified. True micronucleus transformants could be distinguished by resistance to 6-methylpurine from macronucleus transformants. In the transformation of the macronucleus, paromomycin (100 μg / ml final concentration) was added about 4 hours after the transformation and the cells were distributed in aliquots of 100 μl on 96-well microtiter plates. The cells were incubated in a humid box at 30 ° C. After 2-3 days, resistant clones could be identified. Positive clones were inoculated into fresh medium with 120 μg / ml paromomycin. By culturing the cells in this high paromomycin concentration was after reached a complete "phenotypic assortment" (Gaertig & Kapler (1999)) for several generations.

By crossing the micronucleus transformants with a B * VI strain, homozygous knockout mutants could be generated ( Bruns & Cassidy-Hanley, Methods in Cell Biology, Volume 62 (1999) 229-240 ).

EXAMPLE 7: Biolistic Transformation (Microparticle Bombardment)

The transformation of Tetrahymena thermophila was carried out by means of biolistic transformation, as in Bruns & Cassidy-Hanley (Methods in Cell Biology, Vol. 62 (1999) 501-512 ); Gaertig et al. (1999) Nature Biotech. 17: 462-465 ) or Cassidy-Hanley et al. ((1997) Genetics 146: 135-147 ) is described. The handling of the Biolistic® PDS-1000 / He Particle Delivery System (BIO-RAD) is described in detail in the corresponding manual.

For the transformation, 6 mg of gold particles (0.6 μm, BIO-RAD) are loaded with 10 μg of linearized plasmid DNA ( Sanford et al. (1991) Biotechniques 3: 3-16 ; Bruns & Cassidy-Hanley (1999) Methods in Cell Biology, Volume 62: 501-512 ).

Preparation of the gold particles: 60 mg of the 0.6 μm gold particles (Biorad) were resuspended in 1 ml of ethanol. For this purpose, the particles were vigorously mixed 3 times for 1-2 minutes on a vortex. Subsequently, the particles were centrifuged for 1 min (10,000 g) and the supernatant was carefully removed with a pipette. The gold particles were resuspended in 1 ml of sterile water and centrifuged as above. This wash was repeated once, the particles resuspended in 1 ml of 50% glycerol and stored in aliquots of 100 μl at -20 ° C.

Preparing the Transformation: The Macrocarrierhalter, Macrocarrier and Stopscreens were stored for several hours in 100% ethanol, the Rupture Disks in isopropanol. A Macrocarrier was then inserted into the MacrocarrierHalter and dried in the air.

Loading the gold particles with DNA: All work was done at 4 ° C. Gold particles, prepared vector, 2.5M CaCl ₂ , 1M spermidine, 70% and 100% ethanol were chilled on ice. 10 μl of the linearized vector DNA (1 μg / ml) was added to 100 μl of prepared gold particles and gently vortexed for 10 sec. Subsequently, only 100 μl of 2.5 M CaCl _{2 were} added, vortexed for 10 seconds and then 40 μl of 1 M spermidine added and gently vortexed for 10 min. After adding 200 μl of 70% ethanol, the particles were vortexed for 1 min and then centrifuged at 10,000 g for 1 min. The pellet was resuspended in 20 μl of 100% ethanol, centrifuged and then resuspended in 35 μl of 100% ethanol.

The prepared particles were carefully placed with a pipette on the center of a Macrocarriers. The Macrocarrierer was then stored in a box with hygroscopic silica gel until transformation.

Transformation: One ml of the prepared cells (see above) was placed in the center of a 10 micron Tris-HCl (pH 7.5) wetted round filter in a Petri dish and in the bottom shelf of the transformation chamber of the Biolistic® PDS-1000 / He Particle Delivery Systems used. The transformation was carried out with the prepared gold particles at a pressure of 900 psi (two 450 psi rupture disks) and a 27 inches Hg vacuum in the transformation chamber. Subsequently, the cells were immediately transferred to an Erlenmeyer flask with 50 ml SPPA medium and incubated at 30 ° C without shaking.

EXAMPLE 8: Restoration Transformation of Tetrahymanol Knock-out Mutants with the Tetracygenyl Cyclase Genomic Plasmid pgTHC

The transformation was carried out analogously to Example 4. Tetrahymanol knock-out mutants (see Example 2) of Tetrahymena thermophila were used for the transformation. The cells were cultivated in SPPA medium with the addition of 10 mg / l cholesterol. After transformation (see above) with the genomic fragment of the tetrahymanol cyclase (s. Fig. 1 ) the cells were in SPPA medium taken up without cholesterol and incubated at 30 ° C without shaking in the Erlenmeyer flask. After 3 h, the cells were transferred in aliquots of 100 μl to 96 well microtiter plates. The cells were incubated in a moist, darkened box at 30 ° C. After about 2-3 days, the first cholesterol-autotrophic clones could be identified. After about 5-7 days, the positive clones were inoculated into fresh medium. By daily inoculation of the cells over a period of about 2 weeks and multiple isolation of single cells, a complete "phenotypic assortment" (Gaertig & Kapler (1999)) was achieved. These clones were cultured for control in SPPA medium supplemented with paromomycin. After about 3-5 days, all cultures died. Ie. the clones had lost their resistance to paromomycin, which is due to the loss of the neo gene by homologous recombination.

EXAMPLE 9: Restoration Transformation of delta-6 desaturase knockout mutants with the delta-6 plasmid pgDES6

The transformation was carried out analogously to Ex. 4. For the transformation, delta-6 desaturase knock-out mutants (see Ex. 5) of Tetrahymena thermophila were used. The cells were cultivated in SPPA medium supplemented with 200 μg / ml borage oil (20-25% GLA, SIGMA). After transformation (see above) with the genomic delta-6 desaturase DNA fragment (s. Fig. 6 ), the cells were taken up in SPPA medium without Borage oil and incubated at 30 ° C without shaking in Erlenmeyer flask. After 3 h, the cells were transferred in aliquots of 100 μl to 96 well microtiter plates. The cells were incubated in a moist, darkened box at 30 ° C. After about 2-3 days, the first GLA autotrophic clones could be identified. After about 5-7 days, the positive clones were inoculated into fresh medium. By daily inoculation of the cells over a period of about 2 weeks and multiple isolation of single cells, a complete "phenotypic assortment" (Gaertig & Kapler (1999)) was achieved. These clones were cultured for control in SPPA medium supplemented with paromomycin. After about 3-5 days died all cultures. Ie. the clones had lost their resistance to paromomycin, which is due to the loss of the neo gene by homologous recombination.

EXAMPLE 10: Restoration Transformation with the plasmid pgTHC coupled with the neomycin gene

The transformation was carried out analogously to Ex. 8. A vector was used which was constructed as follows:

The genomic fragment of tetra-methanol cyclase (pgTHC) was cut with the restriction enzyme Bgl II. This enzyme cleaves the genomic fragment outside of the coding exons at position 4537 in the 3 'untranslated region. After incubation with T4 DNA polymerase to blunt the ends, the neomycin cassette was ligated into the plasmid. For this, the plasmid p4T2-1ΔH3 was cut with Eco RV / Sma I and the approximately 1.4 kb fragment comprising the neo-cassette was inserted into the already cut plasmid pgTHC into the 3 'untranslated sequence of the Tetrahymena triterpenoid cyclase ligated.

This construct (pgTHC + neo, s. Fig. 4 ) was used for the transformation of the tetrahymanol mutants. After transformation (see above), the cells were taken up in SPPA medium without cholesterol and incubated at 30 ° C. without shaking in the Erlenmeyer flask. After 3 h, paromomycin was added and the cells transferred in 100 μl aliquots to 96-well microtiter plates. The cells were incubated in a moist, darkened box at 30 ° C. After about 2-3 days, the first cholesterol-autotrophic clones were identified that were simultaneously paromomycin-resistant. The positive clones were inoculated into fresh medium without the addition of cholesterol or paromomycin. By daily inoculation of the cells over a period of about 2 weeks and multiple isolation of single cells, a complete "phenotypic assortment" (Gaertig & Kapler (1999)) was achieved. These cells showed good growth even after adding paromomycin.

EXAMPLE 11: Restoration Transformation with the plasmid pgDES6 coupled with the neomycin gene

The transformation was carried out analogously to Ex. 9. A vector was used which was constructed as follows:

The genomic fragment of delta-6-desaturase (pgDES6) was cut with the restriction enzyme Sna BI. This enzyme cleaves the genomic fragment outside of the coding exons at position 747 in the 5 'untranslated region. The plasmid p4T2-1ΔH3 was cut with Eco RV / Sma I and the approximately 1.4 kb fragment comprising the neo-cassette was inserted into the already cut plasmid pgDES6 into the 5'-untranslated sequence of Tetrahymena delta-6 desaturase ligated.

This construct (pgDES6 + neo, s. Fig. 7 ) was used for the transformation of the delta-6 desaturase mutants. After transformation (see above), the cells were taken up in SPPA medium without Borage oil and incubated at 30 ° C without shaking in the Erlenmeyer flask. After 3 h, paromomycin was added and the cells transferred in 100 μl aliquots to 96-well microtiter plates. The cells were incubated in a moist, darkened box at 30 ° C. After about 2-3 days, the first cholesterol-autotrophic clones were identified that were simultaneously paromomycin-resistant. The positive clones were inoculated into fresh medium without the addition of cholesterol or paromomycin. By daily inoculation of the cells over a period of about 2 weeks and multiple isolation of single cells, a complete "phenotypic assortment" (Gaertig & Kapler (1999)) was achieved. These cells showed good growth even after adding paromomycin.

Claims (7)

1. Method for production of recombinant ciliates, comprising the steps:

   a) production of an auxotrophic mutant of the ciliate,

   b) transformation of the mutant with recombinant DNA, containing at least one gene for complementation of the corresponding auxotrophy,

   c) selection of the recombinant ciliates on a minimal medium that permits growth only of the corresponding complemented ciliates,

   wherein ciliates of the genera Paramecium and Tetrahymena are used.
2. Method according to claim 1, in which production of the auxotrophic mutants occurs by knockout of an essential gene.
3. Method according to claim 2, in which the knocked out gene codes for a triterpenoid-cyclase, a delta-6-desaturase or a delta-9-desahirase,
4. Method according to anyone of the claims 1-3, in which the ciliates belong to the genera Paramecium or Tetrahymena, preferably to the species Tetrahymena thermophila.
5. Method according to anyone of the claims 1-4, in which the recombinant DNA for transformation of the auxotrophic ciliate mutant contains a gene for a triterpenoid-cyclase, a delta-6-desaturase or a delta-9-desaturase.
6. Method for production of recombinant proteins, comprising the stages:

   a) production of recombinant ciliates according to the method of anyone of the claims 1-5, wherein the recombinant DNA for transformation of the ciliates additionally contains at least one functional recombinant gene for a protein, which is to be expressed,

   b) culturing of the recombinant ciliates and expression of the proteins,

   c) isolation of the proteins.
7. Protein production method according to claim 6, wherein the recombinant gene for the protein to be expressed was isolated from a vertebrate, preferably from a human.

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